Monoplex PCR Research Articles

Detection of community-acquired respiratory viruses during COVID-19 pandemic in subtropical region in Japan.

Coronavirus disease 2019 (COVID-19) pandemic changed the dynamics of other community-acquired respiratory viruses (CARVs), however, information regarding the frequency of CARV detection during COVID-19 pandemic in subtropical regions is limited. Additionally, studies comparing the diagnostic accuracy between multiplex and monoplex PCR for the diagnosis of COVID-19 are scarce. We evaluated samples collected from patients suspected of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Okinawa, a subtropical climate in Japan, from March 2020 to March 2021. For SARS-CoV-2, results obtained from monoplex (NIID method) and multiplex (Allplex™ SARS-CoV-2 Assay) PCR were compared. In total, 744 samples were collected and 238 viruses were detected in 205 specimens, of which 22 showed viral co-infection. Viral co-infection was more common in patients aged 10 and under than in older patients (p = 0.0054). For CARV, 142 viruses were detected in 127 specimens (17.1%), and human rhinovirus (HRV) was most common. Overall concordance rate for two SARS-CoV-2 assays was 94.1%; 7 and 37 specimens were detected only by NIID and Allplex™ SARS-CoV-2 Assay, respectively. The median cycling threshold values of the 44 samples that were only positive for either the NIID or Allplex™ SARS-CoV-2 Assay were 37.91 for E gene, 38.13 for RdRp/S gene, 38.21 for N gene and 39.16 for N2 gene. HRV was continuously detected during COVID-19 pandemic in the subtropical region and viral co-infection was more common in younger patients. For the diagnosis of COVID-19, multiplex PCR was more reliable, especially in samples with low viral load.

Presence of extended-spectrum beta-lactamase-producing Escherichia coli and carbapenem resistance in ready-to-eat stuffed mussels in Istanbul

AbstractFoodborne pathogens' transmission is essential in the spread of antibiotic resistance, and extended-spectrum beta-lactamase-producing Escherichia coli especially threatens public health. E. coli plays an essential role in the resistance to commonly used beta-lactam group antibiotics. Ready-to-eat (RTE) stuffed mussels are among many restaurants and street vendors, presenting potential health risks of food hygiene origin. 200 RTE stuffed mussels were collected from the Asian and European sides of Istanbul and analysed for the presence of E. coli. As a result of PCR analysis, E. coli was detected in 7 (3.5%) samples. An antibiotic susceptibility test was performed using the disc diffusion method to determine ESBL and carbapenem resistance. All isolates were resistant to ampicillin. The double-disk synergy test was performed as an ESBL phenotypic confirmation test, and no phenotypically ESBL-producing E. coli were detected. The blaTEM gene was detected in one isolate (14.2%) by mPCR, but blaCTX-M, blaSHV, and blaOXA genes were not observed. Meropenem and imipenem were used with the disk diffusion method for carbapenem resistance study, and no resistant isolate was found. Carbapenem resistance genes were investigated by monoplex PCR, and blaNDM-1, blaOXA-48, blaVIM, and blaIMP resistance genes were not detected. This is the first report on ESBL-producing E. coli in RTE stuffed mussels in Türkiye, which draws attention to a public health risk.

Evaluating the efficiency of ELISA, monoplex and multiplex probe-based real-time reverse-transcription PCR assays in the detection of SARS-CoV-2 (COVID-19) and influenza A and B viruses: A cross-sectional study.

The current study aimed to evaluate the efficiency of Enzyme-linked immunosorbent assay (ELISA) assay and monoplex and multiplex real-time reverse-transcription PCR (rRT-PCR) in the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A and B viruses (Flu A and Flu B). The SARS-CoV-2 -specific IgG and IgM antibodies, as well as, Flu A (H1N1 and H3N2 serotypes) and Flu B virus antibodies were determined by ELISA assay. The one-step qRT-PCR method was used to detect the SARS-CoV-2 in nasopharyngeal swab samples. Furthermore, the presence of Flu A and B viruses was evaluated using probe-based RT-PCR. Simultaneous detection of SARS-CoV-2, Flu A and B viruses was performed by multiplex rRT-PCR assay. SARS CoV-2 IgM and IgG antibodies were detected in 33.3% and 58.3% of patients, respectively. In contrast, the SARS CoV-2 genome was detected in 50% of patients using the one-step monoplex RT-PCR assay. Flu A serotypes H1N1 and H3N2 were found in 16.7% and 8.3% of patients. Probe-based RT-PCR revealed that 39.3% of patients were positive for the Flu A virus. Multiplex rRT-PCR detect the SARS-CoV-2, Flu A, and Flu B in 50%, 39.3%, and 19% of samples, respectively. The sensitivity and specificity of multiplex rRT-PCR assay in comparison to monoplex RT-PCR were 100% and 55%, respectively. Coinfection with SARS-CoV-2, Flu A, and Flu B viruses was found in 9.5% of patients. Multiplex rRT-PCR can be used as a repaid, cost-effective and suitable tool for molecular surveillance of SARS-CoV-2 and Flu A/B viruses.

High carriage of plasmid-mediated quinolone resistance (PMQR) genes by ESBL-producing and fluoroquinolone-resistant Escherichia coli recovered from animal waste dumps.

There has been a rise in the consumption of fluoroquinolones in human and veterinary medicine recently. This has contributed to the rising incidence of quinolone resistance in bacteria. This study aimed at the determination of the antibiotic resistance profile of ESBL-producing and fluoroquinolone-resistant E. coli (FQEC) isolated from animal waste obtained from the waste dumps of an agricultural farm and their carriage of genes encoding PMQR. Isolation of ESBL-producing E. coli from animal waste samples was done on CHROMagar ESBL, while presumptive isolates were purified, and identified via the detection of uidA gene. Susceptibility to a panel of ten antibiotics was done using the disc diffusion method, and detection of PMQR genes (qnrA, qnrB, qnrS, aac(6')-lb-cr, qepA and oqxAB) was done using monoplex and duplex PCR. Twenty-five ESBL-producing and FQEC were obtained from the cattle (6), piggery (7) and poultry (12) waste dumps of the farm. There was 100% resistance to cefpodoxime, cefotaxime, enrofloxacin, trimethoprim-sulfamethoxazole and penicillin by the isolates. The resistance to the other antibiotics was streptomycin (48%), ceftazidime (24%), while no isolate resisted amoxicillin-clavulanate and imipenem. The frequencies of PMQR genes detected were; qnrA (96%), oqxAB (96%), qnrB (92%), while qnrS was detected in 88% (22) of the isolates. Aminoglycoside acetyltransferase (aac(6')-lb-cr) and quinolone efflux pump (qepA) were each detected in 20 (80%) of the isolates. This study showed that animal wastes disposed indiscriminately into dumps could be a budding 'hotspot' for multidrug resistant, ESBL-producing and fluoroquinolone-resistant E. coli carrying multiple genes encoding resistance to fluoroquinolone antibiotics.

Standardisation of an In-house Multiplex PCR Assay for Identifying Diffusely Adherent E. coli and ESBL Producing Resistant Isolates

We aimed to develop a multiplex PCR (mPCR) assay utilizing the afaC gene for the detection of DAEC pathotypes and the blaTEM gene for the identification of ESBL production in clinical E. coli isolates along with an Internal Amplification Control (IAC) to rule out false negative test results. Monoplex PCR assays were first established for the afaC gene and blaTEM gene using 60 characterized E. coli isolates from various clinical samples. Subsequently, an mPCR assay was designed to detect both the genes simultaneously along with an IAC to rule out false negative reactions. The effectiveness of this assay was validated using 80 additional clinical isolates. The overall occurrence of DAEC in the study was found to be 0.7% (1/140). ESBL production was detected in 40.7% of the tested isolates, indicating a concerning prevalence of drug-resistant strains. This study emphasizes the value of an in-house mPCR assay as a crucial tool for simultaneously identifying DAEC and ESBL E. coli strains. The inclusion of an IAC in the PCR protocol bolstered the assay’s reliability. This innovation offers a vital resource for effective infection management and contributes to the comprehension of pathogenicity and resistance mechanisms in clinical E. coli isolates.

Dissemination of Integron Class 1 among XDR isolates of P. aeruginosa in Najaf City

Background: Integrons are gene capture systems observed in chromosomes, plasmids, and transposons, where identified and capture multiple gene cassettes. Integerons are consists of 3 elements: the initial is integrase gene (int1) encoding integrase enzyme (int1), which is essential to insertion or deletion of specific site. The second element is attI as integron-connected recombination site. The 3rd element is promoter (Pc), which is required in the expression of cassette-connected genes. 
 Aim of the Study: This thesis designed to examine the occurrence of integron class one genes among XDR isolates of P. aeruginosa. 
 Methods: An overall of 79 specimens were isolated from patients during the period of study and the specimens were carried to the laboratory. By monoplex PCR assay, the 19 XDR isolates of P. aeruginosa were investigated for the presence of integrons class one using intI1primer. 
 Results: This result found that 8 isolates are isolated from burns, 7 isolates are from urine, 4 isolates are from wounds. The PCR results show that 17 (89.5%) of the isolates harbored integrase (intI1) gene, while two isolate (10.5%) was negative for intI1 gene. Conclusions: Present result showed elevated occurrence of integrons class one among XDR isolates of P.aeruginosa.

Detection of virulence genes and multidrug resistance in Pseudomonas aeruginosa clinical isolates from a public hospital in Lagos, Nigeria

Pseudomonas aeruginosa is a main cause of nosocomial infections and is classified as a priority critical pathogen of global concern. Multidrug resistance and virulence genes attributed to the pathogenicity of the organism has resulted in failed therapies, linked to increased morbidity and mortality in infected patients. The aim of this study was to determine the virulence genes and multidrug resistant P.aeruginosa in clinical specimens from a secondary health care center laboratory in Lagos, Nigeria. MethodologyA total of three hundred and sixty-seven clinical samples were examined with conventional methods and Microbact GNB12A. Forty- seven (47) P. aeruginosa strains were identified. Antimicrobial susceptibility test was performed by the standard disk diffusion method following CLSI guidelines. Identification of P. aeruginosa was done by amplification of oprL and oprI genes. Monoplex and multiplex PCR assays were used for detecting virulence genes (lasB, toxA, exoU and nan1). Descriptive statistical methods were used to analyze the distribution of P. aeruginosa by sample sources, MDR patterns by number of isolates and virulence gene carriage by MDR (≥3classes of antibiotics). ResultsA total of forty-seven (47) isolates were confirmed as P. aeruginosa. The isolates showed pan-resistance to cefuroxime and ceftazidime. An elevated level of resistance was also seen in amoxycillin-clavulanic acid, ceftriaxone, cefixime and meropenem. Susceptibility to Amikacin was 95.7 %. Multidrug resistance was seen in forty (85.1 %) of the isolates. P. aeruginosa (100 %) were confirmed with oprL and 74.5 % oprI genes. All isolates (100 %) had lasB, toxA and exoS virulence genes while nan1 was detected in 19.1 % isolates. ConclusionsDiverse and multiple virulence genes were distributed among the multidrug resistant isolates. Understanding the factors contributing to pathogenicity and antibiotic resistance will aid in the treatment of infection. It will also promote judicious use of antimicrobials.

Association of Genetic Variants rs1888747 and rs10868025 of FRMD3 Gene with Diabetic Nephropathy in Pakistani Patients

Background: Diabetic Nephropathy is also known as diabetic kidney disease. The major cause of diabetic nephropathy is diabetes Many candidate genes and genome-wide association studies have proved that gene FRMD3 is highly associated with diabetic nephropathy. Aim of the study: This study aimed to investigate the association of rs1888746 and rs1888747 with diabetic nephropathy in our local population of Pakistan. Research Design: For this study, we enrolled 50 physician-diagnosed patients and 25 healthy control from the young population. Genomic DNA was extracted by organic method and visualized on Agarose gel. Methodology: Monoplex PCR, real-time PCR, genotyping via high-resolution melting curve analysis,and SPSS software were used to accomplish the research work. Results: Our study found that variant rs1888746 shows an insignificant association with (p<0.0982) and rs188747 shows a significant association with (p<0.0498), but results need to be confirmed on a larger scale. Conclusion: The results concluded that our selected rs1888747 on FRMD3 gene shows the allelic association with diabetic nephropathy in Pakistani local population whereas r1888746 did not. Keywords: Genome Wide Association Studies, Diabetic Nephropathy, Genomic DNA, FRMD3 gene

Proinflammatory Cytokines Associated with InvA and PagC Genes of Salmonella typhi Isolated from Patients Undergoing Cholecystectomy

Objective: Cholecystectomy is the surgical removal of the gallbladder. The gallbladder is often colonized by Salmonella during typhoid fever. The association between cytokine and virulence genes of Salmonella is not well known. The current study aims to identify the association between proinflammatory cytokines, TNF-α and IFN-γ, and two virulence genes, InvA and PagC, extracted from S. typhi isolates obtained from patients undergoing cholecystectomy.
 Materials and Methods: One hundred and fifty clinical specimens, including gallbladder tissues and blood, were collected from patients undergoing cholecystectomy at AL-Sadder Medical City and AL-Furat General Hospital/Iraq from December 2019 to September 2020. A Monoplex PCR technique was used to detect InvA and PagC, and an enzyme-linked immunosorbent assay was used to measure serum levels of TNF-α and IFNγ.
 Results: From a total of 66 S. typhi isolates, the prevalence of InvA and PagC was (84.8% and 69.6%) respectively. Even after adjusting for potential confounding factors, serum levels of TNF-α and INF-γ were statistically significantly higher in PagC detected in S. typhi isolates compared to PagC not detected (p-value<0.001), but there was no significant difference between InvA detected in S. typhi isolates and InvA not detected (p-value > 0.05).
 Conclusion: The virulence genes of S. typhi, especially PagC, may be considered a potent inducer of proinflammatory cytokine secretion. Further studies are needed to present the patient‘s additional clinical course according to cytokine level.

Identification of commensal Pseudomonas aeruginosa isolates using duplex PCR targeting the oprL and algD genes

Background: Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous bacterium which can be found in most moisture places such as soil, water, food, plants, and animals including humans. Due to genetic flexibility among strains, there is no standard molecular identification for P. aeruginosa from different sources, particularly for human colonizing P. aeruginosa or commensal isolates. Materials and Method: Monoplex PCR using published primers of oprL, algD, nfxB gene were compared for their specificity and sensitivity in identification of commensal P. aeruginosa. Twenty- nine human colonizing Pseudomonas isolates including 16 P. aeruginosa isolates and 13 P. aeruginosa-like isolates were used in the study. To improve the detection, two new primer pairs targeting oprL gene was designed (oprL-pp1 and oprL-pp2), and oprL-algD duplex PCR was carried out with newly designed oprL primer pairs. Result and conclusion: PCR targeting algD or nfxB genes has the same sensitivity of 93.75% and specificity of 100%, while oprL-specific PCR using published primers is more sensitive (100%) but less specific (0%). Duplex PCR yielded high sensitivity and specificity in detecting P. aeruginosa. Both oprL-pp1/algD and oprL-pp2/algD duplex-PCR had 93.75% sensitivity (15/16 P. aeruginosa isolates) and 100% specificity (0/13 P. aeruginosa-like isolates). Besides, oprL-pp2 primers were more specific than oprL-pp1 primers, with only 2 over 13 P. aeruginosa-like isolates detected, while oprL-pp1 primers detected all P. aeruginosa-like isolates. Compared to the monoplex PCR that only targeted oprL gene, the duplex-PCR utilizing oprL-pp2 and algD primer can identify 15/16 P. aeruginosa isolates (93.75% specificity) and reduce the number of oprL-positive isolates required further exact identification. Additionally, the duplex-PCR used in this study was negative for non-Pseudomonas species including E. coli, V. cholera, V. parahaemolyticus, S. aureus. In conclusion, our duplex PCR targeting oprL and algD could be a valuable tool for P. aeruginosa screening.

Genetic analysis and antibiotic susceptibility of Serratia fonticola isolated from ornamental birds in Iraq

The current study was aimed to detect the prevalence of Serratia fonticola strains in birds existed at birdmarkets and houses in different areas of Iraq. One hundred fifty Samples were collected from the wounds and tracheal swab in birds. Samples were cultured on MacConkey and blood agar. Identification of isolate was made according to colony morphology, Gram staining, and conventional biochemical interactions and analysis profile Vitek® 2 compact system. The culture results were revealed an overall one positive isolate with detection an antimicrobial susceptibility test by Vitek® 2 compact system and isolate showed that was susceptible to (Cefepime, Ceftazidime, Amikacin and Ciprofloxacin). In contrast, absolute resistant against (Aztreonam, Gentamicin and Trimethoprim/Sulfamethoxazole). Positive culture isolate was confirmed as Serratia fonticola by monoplex PCR assay, targeting 16S rRNA gene. Positive DNA product were sequenced, and recorded in the NCBI under the accession number (MN696514.1) for birds. The NCBI-BLAST Homology sequence identity reported a significant similarity between these local Serratia fonticola isolates and NCBI isolates; South Korean ( MK235160.1), Egyptian (MN865872.1), Russians (LT555292.1) Iranian (MK737094.1) isolate, at 99%. For our knowledge, this is the first molecular reports performed in Iraq for detection Serratia fonticola infections in birds in Iraq.

Clinical carbapenem-resistant Klebsiella pneumoniae isolates simultaneously harboring bla NDM-1, bla OXA types and qnrS genes from the Kingdom of Bahrain: Resistance profile and genetic environment.

The prevalence of Carbapenem-resistant Klebsiella pneumoniae (CRKP) is currently increasing worldwide, prompting WHO to classify it as an urgent public health threat. CRKP is considered a difficult to treat organism owing to limited therapeutic options. In this study, a total of 24 CRKP clinical isolates were randomly collected from Salmaniya Medical Complex, Bahrain. Bacterial identification and antibiotic susceptibility testing were performed, on MALDI-TOF and VITEK-2 compact, respectively. The isolates were screened for carbapenem resistance markers (bla NDM, bla OXA-23, bla OXA-48 and bla OXA-51) and plasmid-mediated quinolone resistance genes (qnrA, qnrB, and qnrS) by monoplex PCR. On the other hand, only colistin-resistant isolates (n=12) were screened for MCR-1, MCR-2 and MCR-3 genes by monoplex PCR. Moreover, the Genetic environment of bla NDM, integrons analysis, and molecular characterization of plasmids was also performed. Antibiotic susceptibility revealed that all the isolates (100%) were resistant to ceftolozane/tazobactam, piperacillin/tazobactam, 96% resistant to ceftazidime, trimethoprim/sulfamethoxazole, 92% resistant to meropenem, gentamicin and cefepime, 88% resistant to ciprofloxacin, imipenem, and 37% resistant to amikacin. Ceftazidime/avibactam showed the least resistance (12%). 75% (n=12/16) were resistant to colistin and 44% (n=7/16) showed intermediate susceptibility to tigecycline. The detection of resistant determinants showed that the majority (95.8%) of CRKP harbored bla NDM-1, followed by bla OXA-48 (91.6%) bla OXA-51 (45.8%), and bla OXA-23 (41.6%). Sequencing of the bla NDM amplicons revealed the presence of bla NDM-1. Alarmingly, 100% of isolates showed the presence of qnrS. These predominant genes were distributed in various combinations wherein the majority were bla NDM-1 + bla OXA-51+ qnrS + bla OXA-48 (n =10, 41.7%), bla NDM-1 + bla OXA-23+ qnrS + bla OXA-48 (n=8, 33.3%), among others. In conclusion, the resistance rate to most antibiotics is very high in our region, including colistin and tigecycline, and the genetic environment of CRKP is complex with the carriage of multiple resistance markers. Resistance to ceftazidime/avibactam is uncommon and hence can be used as a valuable option for empirical therapy. Molecular data on resistance markers and the genetic environment of CRKP is lacking from this geographical region; this would be the first report addressing the subject matter. Surveillance and strict infection control strategies should be reinforced in clinical settings to curb the emergence and spread of such isolates.

Validation of a duplex PCR technique using the gen E and RNase P for the diagnosis of SARS-CoV-2

IntroductionReverse transcriptase - polymerase chain reaction (RT-PCR) is the standard technique for SARS-CoV-2 diagnosis. The World Health Organization recommends the Charité-Berlin protocol for COVID-19 diagnosis, which requires triple PCR, limiting the process capability of laboratories and delaying the results. In order to reduce these limitations, a duplex PCR is validated for the detection of the E and ribonuclease P genes. MethodsWe compared the limit of detection, sensitivity and specificity of the duplex PCR technique (E gene and Rnasa P) against the monoplex standard (E gene) in RNA samples from a SARS-CoV-2 isolate and 88 clinical specimens with previously known results. The repeatability and reproducibility of the threshold cycle values ​​(Ct) were determined in two independent laboratories of the Faculty of Medicine of the Universidad de Antioquia, using different reagents and real time instruments. ResultsThere were no significant differences in the Ct results between both techniques (P = .84). Using the monoplex PCR of E gene as a reference, the interrater reliability analysis showed similarity between the two techniques, with a kappa coefficient of 0.89, the sensitivity and the specificity of duplex PCR were 90% and 87%, respectively. ConclusionsDuplex PCR does not affect the sensitivity and specificity reported by the Charité, Berlin protocol, being a useful tool for SARS-CoV-2 screening in clinical samples.

MOLECULAR DETECTION OF THE TWO VIRULENCE GENES HWP1 AND ALS1 IN CANDIDA SPECIES ISOLATED FROM ONYCHOMYCOSIS

The aim: This article was aimed to assess the prevalence of Candida species causing nail infections and to investigate the most important virulence genes that cause adhesion. Materials and methods: Samples were collected from General Hospital and the outpatient's clinics in Thi-Qar Province, southern Iraq, during the period from January 2021 to May 2021. Samples were inoculated on Sabouraud Dextrose Agar plates containing 0.5 mg chloramphenicol, then Petri dishes were incubated at 30°C under aerobic conditions and observed for Candida species growth for 24-72 hrs. All isolates were identified by morphologic and chromatic properties on CHROMagar and by Api Candida. Results: Twenty five isolates were obtained from hundred samples, which belong to five species of the genus Candida, distributed in percentage 9(36%), 4(16%), 4(16%), 4(16%) and 3(12%) of C. glabrata, C. krusei, C. albicans, C. famata and C. tropicalis consecutively and one isolate was not identified. Monoplex PCR technique was used to detect virulence genes (hwp1 and als1) of Candida species. Four Candida albicans isolates (16%) were carried of both hwp1 and als1 genes. Conclusions: The virulence gene (hwp1) can be relied upon in molecular detection as a special diagnostic tool of Candida albicans species except other types causing Candidal onychomycosis.

Assessment of a multiplex RT-PCR for Simultaneous, Rapid Screening of Common Viral Infections of Central Nervous System: A Prospective Study for Enteroviruses and Herpesviruses

Abstract Introduction Acute meningitis is a common neurological disorder that affects both children and adults and has a high mortality rate. This study aimed to create a multiplex reverse transcriptase PCR system for screening clinical samples for the presence of the two viruses currently considered to be the most common causes of acute meningitis in Asia. Materials and Methods A single-tube RT multiplex PCR assay was developed and tested for sensitivity and specificity using primers that have been commonly used to screen for herpes simplex viruses 1 and 2 (HSV-1/2) and enterovirus (EV) in clinical samples. The procedure was then used to screen 303 clinical samples for the target viruses, which included 101 feces samples, 101 throat swabs, and 101 cerebrospinal fluid (CSF) samples obtained from 101 hospitalized Iranian children with suspected viral meningitis/meningoencephalitis, and the findings were compared to those of an RT monoplex PCR method. Results The RT-PCR approach demonstrated high precision, with no non-target virus amplification. The results of using this assay to screen clinical samples revealed that RT monoplex PCR had the same sensitivity as RT multiplex PCR for the three different types of specimens. Conclusions This newly developed multiplex RT-PCR method is a simple, fast diagnostic tool that can be used to screen clinical samples for viruses that cause acute meningitis/meningoencephalitis in children.

Short Communication: Molecular study of bacteria isolated from meat and chicken frozen from Misan Governorate market in Iraq

Abstract. Husain DA, Aziz ZS. 2021. Short Communication: Molecular study of bacteria isolated from meat and chicken frozen from Misan Governorate market in Iraq. Biodiversitas 23: 81-86. Food contamination is frequently defined as spoiled or tainted foods because they contain microorganisms, such as fungi, bacteria, parasites, or toxic substances that make them unfit for consumption. In this study, one hundred meat and chicken Frozen products samples were collected randomly from the commercial markets in Misan Governorate, Iraq from November 2020 to April 2021, included: (burger – sausage – kebab – shawarma – minced meat) of meat and (chest-thigh-liver-burger-kebab) of Chicken. The results of aerobic plate count showed that all meat and chicken products were contaminated with bacteria, but the imported products were more contaminated than the local products. Our study included the isolation and diagnosis of eighteen bacterial species by using routine and standard bacteriological tests. The results showed that Gram-negative bacteria were common in meat products (52%) and Gram-positive bacteria represented 48%. In chicken samples, the Gram-positive bacteria was most dominant (59%) while the percentage of Gram-negative bacteria was 41%. Monoplex PCR was used to identify eighteen bacterial strains using a universal 16S rRNA primer that gave 1500 bp amplification product, nucleotide sequences were studied at the National Center for Biotechnology Information (NCBI) using the Basic Local Alignment Search Tool (Nucleotide BLAST). The diagnosed bacteria were Aeromonas veronii, Pseudomonas plecoglossicida, Acinetobacter lwofii, Aeromonas veronii, Klebsiella pneumoniae, Pseudomonas japonica, Pseudomonas songnenensis, Klebsiella pneumoniae subsp. ozaenae, Psychrobacter sanguinis, Klebsiella pneumoniae strain, Acinetobacter lwofii, Lysinibacillus boronitolerans, Bacillus licheniformis, Enterobacter hormaechei, Pseudomonas putida strain, Serratia liquefaciens strain, Comamonas testosterone strain, and Methylogaea oryzae strain which recorded in the Gene Bank belong to different accession numbers.

Rapid and specific detection of Mycoplasma gallisepticum and Mycoplasma synoviae infection in poultry using single and duplex PCR assays

Avian mycoplasmosis, mainly caused by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), is an economically important disease of poultry industry. The present study was aimed to develop duplex PCR as a rapid, specific and economical method for accurate detection of MG and MS in poultry and its comparison with single (monoplex) MG/MS PCR. During present investigation, a total of 146 poultry flocks having clinical history of respiratory disease were screened. Pooled tissue samples (trachea, lungs and air sacs) from 4-5 birds of each flock were collected during necropsy at disease investigation laboratories, Hisar, Haryana, India. The single and duplex PCR assays were standardized using primers of intergenic spacer region (IGSR; 16S-23S rRNA) for MG and hemagglutinin vlhA gene for MS, with expected amplicon size of 812 bp and 1200 bp products, respectively. In single PCR, 6.85%, 2.74% and 2.74% tissue samples were found positive for MG, MS and both MG and MS, respectively. However, duplex PCR showed, 7.53%, 2.74% and 1.37% positivity for MG, MS and both MG and MS, respectively. Taking the results of monoplex PCR as a gold standard, sensitivity and specificity of the developed duplex PCR was found to be 94.44% and 100%, respectively. Moreover, Cohen's kappa statistic (k = 0.97) measured a ‘perfect’ agreement between monoplex and duplex PCR assays. The positive and negative predictive values of duplex PCR was found to be 1.0 and 0.9922, respectively at 95% confidence interval (CI), as compared to monoplex PCR. The simultaneous use of two genes in a duplex PCR was more rapid and economical than two separate single PCR reactions.

Genotypic and Phenotypic Characterizations of Methicillin-Resistant Staphylococcus aureus (MRSA) on Frequently Touched Sites from Public Hospitals in South Africa.

The hospital environment acts as a reservoir in the transmission of pathogens, such as MRSA, which may cause hospital-acquired infections. This study aimed to ascertain the prevalence, genetic relatedness, antibiotic resistance, and virulence profile of MRSA on some frequently touched hospital sites in South Africa. A total of 777 swabs were randomly collected from 11 frequently touched sites in the hospital environment of three wards of four public hospitals in the KwaZulu-Natal province of South Africa. Isolation of S. aureus and confirmation were done using genotypic and phenotypic methods. Antibiotic susceptibility testing was performed using the Kirby–Bauer disk-diffusion method. MRSA isolates were determined by the presence of the mecA gene. Virulence and resistance genes were detected using a standard monoplex PCR assay. ERIC-PCR was conducted to evaluate the genetic relatedness. An overall prevalence of 12.7% for S. aureus isolates was obtained. Out of these, 89.9% (89/99) were confirmed to be MRSA. The sites with the highest prevalence were the occupied beds (16.2% (16/99)), unoccupied beds (16.2% (16/99)), patient files (14.1% (14/99)), ward phones (13.1% (13/99)), and nurses' tables (14.1% (14/99)). The virulence genes with the highest observed frequency were hld (87 (87.9%)) and LukS/F-PV (53 (53.5%)). The resistance genes with the highest frequency were the tetM and tetK genes detected in 60 (60.6%) and 57 (57.6%) isolates, respectively. The ERIC-PCR results obtained indicated a high level of genetic diversity; however, intraclonal (within a hospital) and interclonal (between hospitals) clusters of MRSA were observed. The study showed that MRSA can contaminate various surfaces, and this persistence allows for the dissemination of bacteria within the hospital environment. This highlights the need for improved infection prevention and control (IPC) strategies in public hospitals in the country to curb their potential transmission risks.

Establishment of a Multiplex RT-PCR Method for the Detection of Five Known Genotypes of Porcine Astroviruses.

Porcine astroviruses (PAstVs) are prevalent in pigs worldwide, and five genotypes have been reported to circulate in China. However, little is known about the coinfection status of PAstVs. For differential and simultaneous diagnoses of these five genotypes of PAstVs, a multiplex RT-PCR method was established on the basis of the ORF2 gene of type 1 PAstV, and the ORF1ab genes of type two to five PAstVs. This quintuple PCR system was developed through optimization of multiplex PCR and detection sensitivity and specificity. The results showed that this multiplex RT-PCR method could specifically detect all the five PAstV genotypes without cross-reaction to any other major viruses circulating in Chinese pig farms. The detection limit of this method was as low as 10 pg of standard plasmids of each PAstV genotype. In addition, a total of 275 fecal samples collected from different districts of Guangxi, China, between April 2019 and November 2020, were tested by this newly established multiplex RT-PCR. Moreover, the sensitivity and specificity of monoplex and multiplex RT-PCR methods were compared by detecting the same set of clinical positive samples. The results revealed that PAstV1 (31/275), PAstV2 (49/275), PAstV3 (36/275), PAstV4 (41/275), and PAstV5 (22/275) were all detected, and dual (PAstV1+PAstV2, PAstV1+PAstV3, PAstV2+PAstV3, PAstV2+PAstV4, PAstV3+PAstV4, and PAstV4+PAstV5) or triple genotypes (PAstV1+PAstV2+PAstV3 and PAstV2+PAstV3+PAstV4) of coinfections were also unveiled in this study. The detection result of multiplex PCR was consistent with that of monoplex PCR. Compared with monoplex PCR, this multiplex PCR method showed obvious advantages such as time and cost efficiency and high sensitivity and specificity. This multiplex RT-PCR method offered a valuable tool for the rapid and accurate detection of PAstV genotypes circulating in pig herds and will facilitate the surveillance of PAstV coinfection status.

Biofilm formation by LEE-negative Shiga Toxin–Producing Escherichia coli strains

Shiga toxin-producing Escherichia coli (STEC) include several serotypes isolated from cases of hemorrhagic colitis and, hemolytic uremic syndrome. Although O157:H7 is the most predominant STEC serotype, more than 100 non-O157 serogroups cause diseases in humans. Some STEC carry a Locus of Enterocyte Effacement (LEE-positive); however, STEC that do not carry LEE (LEE-negative) have also been associated with illness, mainly those harbouring the Locus of Adhesion and Autoaggregation (LAA). LAA carry some genes such as hes, iha, tpsA, and agn43, related with pathogenicity. One of them is the ability to form biofilms on different environments, which can contaminate food and generate infections while protecting themselves against adverse conditions. Considering that LAA could be responsible for some adherence mechanisms, the aims of this study were to compare different serogroup of LEE-negative STEC strains in their ability to form biofilms and to evaluate the participation of some genes encoding in LAA. A total of 348 LEE-negative STEC strains was analyzed. The presence of hes, iha, tpsA and agn43 were determined by monoplex PCR. From them, 48 STEC strains belonging to serogroups O113, O130, O171, O174 and, O178 were assayed for their ability to form biofilm. The most prevalent genes detected were agn43 (72.1%) and tpsA (69.5%). The iha and hes genes were present in 63.7% and 54% of the strains, respectively. Although all STEC strains were able to form biofilm, it was found a high variability between them. The relation between the biofilm formation and the presence of each gene was not statistically significant, suggesting that biofilm formation is independent of the presence of those genes. Highlighting that there is no treatment for HUS, it is once again notable that prevention measures and control strategies to prevent biofilm formation are important factors in reducing STEC transmission.